This invention relates to liquid chromatography (xe2x80x9cLCxe2x80x9d) and, particularly, to fraction collection in liquid chromatography.
Background Compounds may be collected as they elute from liquid chromatographic columns (xe2x80x9cLC columnsxe2x80x9d) and then subjected to further analysis as, for example, by mass spectrometry (xe2x80x9cMSxe2x80x9d). For complex mixtures, the chromatographic column may be coupled directly with a mass spectrometer or other analytical apparatus. Where LC and MS are carried out as a unified, continuous process, it is known as liquid chromatography/mass spectrometry (xe2x80x9cLC/MSxe2x80x9d).
Typically the effluent from the LC column is in a highly dilute liquid phase, with the separated components emerging from the LC column as xe2x80x9cpeaksxe2x80x9d entrained within a liquid carrier. This can present significant problems for efficiently coupling the effluent with the ion source of the mass spectrometer. Nonvolatile inorganic components present in the eluent, for example as buffers, can interfere with ionization, and where gradient elution is employed the composition of the solvent changes in the course of the analysis. Typical flow rates of LC eluents produce, after vaporization, the equivalent of gas flows much too high for conventional ion sources to accommodate. The difficulty of removing the liquid solvent from the components to be analyzed in the mass spectrometer has raised significant challenges for the development of LC/MS systems.
Generally, in a conventional LC/MS analysis, the sample to be analyzed is separated using an LC column, and the eluent is directed to the mass spectrometer, which completely consumes the sample stream. Optionally, the LC eluent may be passed through a nondestructive detector, such as an optical detector, to provide general information as to the presence of peaks in the flow, without destroying the sample in the stream, before the sample stream is sent on to the MS. Optical detectors conventionally include, for example, UV-Vis, Refractive Index, and fluorescence detectors.
It may be desirable to employ a fraction collection system, to physically collect a purified sample or samples from a mixture in the LC eluent stream. In one conventional approach, a nondestructive detector is employed upstream from the fraction collector, and the time delay between the moment a peak is detected at the detector and the time of arrival of the peak at the fraction collector is calculated from the known or estimated flow rate and the measured or calculated volume capacity of the tubing and connections between the detector and the fraction collector. This calculated time delay provides a basis for an expectation of when a component of the sample, detected at the nondestructive detector, should arrive at the collection vessel. The sample component is presumed to be contained in a collection vessel that received the eluent flow at the estimated arrival time, and the material in that collection vessel can be selected for further analysis.
In one approach to fraction collection, the eluent stream is split so that the sample stream can be subject to destructive analysis without consuming the sample. In a mass-based fraction collection system, for example, a portion of the sample stream is sent to a mass detector to detect the presence of a sample, identifiable by specific ions or ion signals, and the rest of the stream is sent to the fraction collector. According to the invention the time a sample component is detected at the destructive detector (MS in a mass-based system) is used to predict when the stream containing that sample component should arrive at the fraction collector, permitting accurate and reliable placement of the purified sample component in an identified collection vessel or further sample stream for further analysis.
In one aspect the invention features fraction collection apparatus including a first conduit connected at its upstream end to the outlet from a liquid chromatography column and connected at its downstream end to the inlet of a flow splitter, a second conduit connected at its upstream end to a first outlet from the flow splitter and connected at its downstream end to a sample collector, and a third conduit connected at its upstream end to a second outlet from the flow splitter and connected at its downstream end to a destructive detector; a first nondestructive detector is configured near the sample collector to detect passage of a sample component in the third conduit and, optionally, a second nondestructive detector is situated upstream from the splitter to detect passage of a sample component in the first conduit.
In some embodiments the destructive detector is a mass spectrometer or an evaporative light scattering detector or an electrochemical detector. In some embodiments each or both of the first and the second non-destructive detector is an optical detector, such as a UV-Vis absorption detector, a fluorescence detector, or a refractive index detector. In some embodiments the conduits comprise tubing or channels formed in a solid substrate; where an optical detector is employed as a nondestructive detector the conduit preferably is constructed of a material that permits transmission of the wavelength (UV, visible) or wavelengths employed in the detection.
In some embodiments the fraction collection apparatus further includes a fourth conduit connected at its upstream end to a third outlet from the flow splitter and connected at its downstream end to a quantitative detector. In still other embodiments having a fourth conduit connected at its downstream end to a quantitative detector, the apparatus further includes a second splitter having an inlet and a first outlet connected inline in either the second conduit or the third conduit, and a second outlet connected to the upstream end of the fourth conduit. In some embodiments the quantitative detector includes an evaporative light-scattering detector or a nitrogen-sulfur detector.
In some embodiments the fraction collection apparatus is operatively connected to automated controls which, in particular embodiments, receive and process data respecting the detection times at the destructive detector and at one or both of the nondestructive detectors. Preferably the automated controls include a computer capable of receiving signals from the detectors and determining intervals between arrival times, and preferably the automated controls are further capable of employing the time interval data dynamically to activate and control the collection of particular fractions.
In another aspect the invention features a method for collecting a sample component in a sample stream, by dividing the sample stream into a first stream passing into a destructive detector and a second stream passing to a sample collector, introducing a calibrant into the sample stream and determining the difference in time TD between the detection of the calibrant at the destructive detector and detection of the calibrant at a nondestructive detector near the sample collector, then detecting the sample component in the first stream using the destructive detector and determining the time T2 the sample component was detected by the destructive detector, and collecting the sample component at a time T3 equivalent to TD+T2.
In still another general aspect the invention features a method for calibrating an expected time of arrival at a collector of a sample component in a sample stream from an LC column, by dividing the sample stream into a first stream passing to a destructive detector and a second stream passing to a sample collector, introducing a calibrant into an eluent stream and determining the difference in time TD between the detection of the calibrant at the destructive detector and detection of the calibrant at a nondestructive detector near the sample collector, whereby a sample component introduced into a similar eluent stream and detected at the destructive detector at a time T2 is expected to arrive at the sample collector at a time TD later than T2.